Mechanistic link between β barrel assembly and the initiation of autotransporter secretion

Pavlova, Olga A.; Peterson, Janine H.; Ieva, Raffaele; Bernstein, Harris

doi:10.1073/pnas.1219076110

Cited by 83 publications

(133 citation statements)

References 42 publications

(75 reference statements)

Supporting

Mentioning

128

Contrasting

Order By: Relevance

“…Conversely, mutational analyses of EspP show delayed passenger domain translocation in the mutant proteins EspPG 1066 A and EspPG 1081 D, that did not affect interactions with the periplasmic chaperone Skp nor the BAM complex 42 . It was concluded that these mutations affect the insertion of the EspP barrel domain into the plane of the outer membrane 42 . Analysis of the crystal structure of EspP shows that these mutations, EspPG 1066 A and EspPG 1081 D, would each affect a mortise-tenon structure (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 88%

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

et al. 2014

View full text Add to dashboard Cite

Bacterial autotransporters comprise a 12-stranded membrane-embedded b-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Here, paired glycine-aromatic 'mortise and tenon' motifs are shown to join neighbouring b-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cells. In line with this, biophysical studies of the autotransporter Pet show that the conserved residues significantly quicken completion of the folding reaction and promote stability of the autotransporter barrel domain. Comparative genomics demonstrate conservation of glycine-aromatic residue pairings through evolution as a previously unrecognized feature of all autotransporter proteins.

show abstract

Section: Discussionmentioning

confidence: 88%

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

et al. 2014

View full text Add to dashboard Cite

show abstract

“…The Bam complex appears to promote the secretion of the passenger domain as well as the membrane insertion of the β-barrel, and the assembly of EspP into proteoliposomes that contain the Bam complex has been reconstituted in vitro (40). The passenger domain is secreted in a C-to N-terminal direction (57,58), and even large N-terminal deletions do not affect protein biogenesis (31,59). Because we wished to focus on the assembly of the EspP β-barrel, we used a ~35 kD truncated version of EspP that lacks most of the passenger domain (EspP∆5, residues 998-1300) in our experiments.…”

Section: The Bam Complex Catalyzes Efficient Folding Of Espp Into a Wmentioning

confidence: 99%

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Hussain

Bernstein

2018

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "b-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively, but often requires nonphysiological conditions and time scales and is strongly influenced by properties of the lipid bilayer including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the b-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA and another model protein (OmpG) folded at significantly different rates and that an a-helix embedded inside the EspP b-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding. __________________________ Gram-negative bacteria, a class that includes many pathogenic and emerging antibiotic-resistant organisms, are bound by a double cell membrane. Most of the proteins that reside in the outer membrane (OM) 2 , which serves as a robust barrier, have a distinctive "b-barrel" architecture. A bbarrel is essentially a b-sheet rolled into a closed cylinder that has a hydrophobic exterior and a hydrophilic interior and that is held together by hydrogen bonds between the first and last bstrands. The b-barrel scaffold is found exclusively in the OM of bacteria and organelles of bacterial origin. OM proteins (OMPs) mediate many different functions vital for bacterial survival and range considerably in size from 8-26 b-strands (1, 2). Some OMPs form oligomers (3), while others contain a segment that is embedded inside the bbarrel or a separately folded domain that is displayed on either the periplasmic or extracellular side of the OM (1).OMPs must first traverse the inner membrane (IM) and the periplasmic space, which is devoid of ATP (4), before they attain their final structure in the OM. After OMPs are transported across the IM in an unfolded conformation through the Sec translocon, a variety of periplasmic chaperones including SurA, Skp, and DegP prevent their aggregation in the periplasm (5-7). Integration into the OM is then catalyzed by the heterooligomeric β-barrel assembly machinery (Bam) complex (8,9). In E. coli, the Bam complex is composed of BamA, a b-barrel protein that contains five http://www.jbc.org/cgi

show abstract

“…Finally, a component of the Bam complex (BamA) has been shown to interact with the passenger domain during the translocation reaction (7). Although the β-domain does appear to play a role in translocation (20,21), available evidence suggests that the Bam complex promotes the membrane insertion of the β-domain and the secretion of the passenger domain in a concerted reaction (7,8).…”

mentioning

confidence: 99%

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

Kang'ethe

Bernstein

2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Autotransporters are a large class of virulence proteins produced by Gram-negative bacteria. They contain an N-terminal extracellular ("passenger") domain that folds into a β-helical structure and a C-terminal β-barrel ("β") domain that anchors the protein to the outer membrane. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the β-helix in the extracellular space facilitates unidirectional secretion of the passenger domain. In this study we used a chimeric protein composed of the 675-residue receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C terminus of the Escherichia coli O157:H7 autotransporter EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety was efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalled translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, together with the finding that naturally occurring passenger domains are predominantly acidic, provide evidence that a net negative charge plays a significant role in driving the translocation reaction.bacterial pathogenesis | membrane protein assembly | type Va secretion

show abstract

Mechanistic link between β barrel assembly and the initiation of autotransporter secretion

Cited by 83 publications

References 42 publications

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

Contact Info

Product

Resources

About